slide 2:

slide 3:

RNAi
 RNAi is a powerful conserved biological process
through which the small double-stranded RNAs
specifically silence the expression of homologous
genes largely through degradation of their cognate
mRNA.
 its responsible for post-transcriptional gene silencing of the
gene from which it was derived
 Endogenous cellular mechanisms
 Effecter molecules for functional genomics
 Great potential as therapeutic agents for treatment
of human disease

slide 4:

RNA interference Technology
 RNAi is used to block the expression of genes and
create phenotypes that can potentially yield clues
about the function of these genes.
 In the post-genomic era the elucidation of the
physiological function of genes has become the rate-
limiting step in the quest to develop ‘gene-based
drugs’ and RNAi could potentially play a pivotal role in
the validation of such novel drugs.
1 http://www.youtube.com/watchvH5udFjWDM3Efeaturerelated
2 http://www.youtube.com/watchvA-l8tqjm4Vgfeaturerelated
3 http://www.youtube.com/watchv3kdhYCJFmZcfeaturerelated
4 http://www.youtube.com/watchvkCxQdXX0Dbk
5 http://www.youtube.com/watchvh1kayIVEfcYfeaturerelated
RNAi is a fantastic discovery all the RNAi idea will be describing in these video

slide 5:

Time -
Line

slide 6:

Discovery of RNAi or
PTGS
Post transcriptional gene
silencing
•Also called Co-suppression
Suppression was mostly
due to increased
degradation of the mRNAs
from the endogenous and
introduced genes
First discovered in plants
R. Jorgensen 1990
•When Jorgensen introduced
a re-engineered gene into
petunia that had a lot of
homology with an
endogenous petunia gene
both genes became
suppressed
Flowers from 3 different transgenic petunia
plants carrying copies of the chimeric DFR
gene above. The flowers had low DFR
mRNA levels in the non-pigmented areas
but gene was still being transcribed.

slide 8:

Nobel Prize in 2006
Field of Physiology
Medicine
-RNAi can be induced in C. elegans in
three simple ways:
-Injection of dsRNA into the worm
gonads
-Soaking the worms in dsRNA solution
-Feeding the worms engineered
bacteria producing dsRNA
RNAi discovered in Nematode Caenorhabditis elegans 1998
first animal while attempting to use antisense RNA in vivo
Craig Mello Andrew Fire
Control “sense” RNAs also produced suppression of target gene
sense RNAs were contaminated with dsRNA.
dsRNA was the suppressing agent.

slide 9:

Double-stranded RNA dsRNA induced interference
of the Mex-3 mRNA in the Nematode
Caenorhabditis elegans
Antisense RNA c or
dsRNA d for the mex-
3 mRNA was injected
into C. elegans
ovaries and then mex-
3 mRNA was detected
in embryos by in situ
hybridization with a
mex-3 probe.
a control embryo
b control embryo hyb.
with mex-3 probe
Conclusions: 1 dsRNA reduced mex-3 mRNA better than antisense
mRNA. 2 the suppressing signal moved from cell to cell.

slide 10:

Mechanism/Process
 A cellular mechanism that degrades unwanted RNAs
in the cytoplasm but not in the nucleus
What happens
 dsRNA is processed into shorter interfering siRNAs
that guide the targeted cleavage of homologous RNA.

slide 12:

A model for the mechanism of RNAi
- Silencing triggers in the form of double-
stranded RNA may be presented in the cell as
synthetic RNAs replicating viruses or may be
transcribed from nuclear genes.
- These are recognized and processed into
small interfering RNAs by Dicer.
- The duplex siRNAs are passed to RISC
RNA-induced silencing complex
- The complex becomes activated by unwinding
of the duplex.
- Activated RISC complexes can regulate gene
expression at many levels:
•Promoting RNA degradation
•Translational inhibition
•Chromatin remodelling
- Amplification of the silencing signal in plants
may be accomplished by siRNAs priming RNA-
directed RNA polymerase RdRP-dependent
synthesis of new dsRNA.

slide 13:

Mechanism of RNA
interference

slide 14:

Mechanism of RNAi : Role of
Dicer
 Cells plants and animals undergoing RNAi
contained small fragments 25 nt of the RNA
being suppressed.
 A nuclease Dicer was purified from Drosophila
embryos that still had small RNA fragments
associated with it both sense and antisense.
 The Dicer gene is found in all organisms that
exhibit RNAi and mutating it inhibits the RNAi
effect. Conclusion: Dicer is the endonuclease that degrades dsRNA
into 21-24 nt fragments and in higher eukaryotes also pulls
the strands apart via intrinsic helicase activity.

slide 17:

Responses to Mechanical Stimuli
17
HIV levels can
be reduced by
30-50 fold by
siRNA

slide 19:

Biotechnology Agriculture
 RNA interference has been used for applications in
biotechnology particularly in the engineering of food plants
that produce lower levels of natural plant toxins. Such
techniques take advantage of the stable and heritable
RNAi phenotype in plant stocks.
 For example cotton seeds are rich in dietary protein but
naturally contain the toxic terpenoid product gossypol
making them unsuitable for human consumption.
 RNAi has been used to produce cotton stocks whose
seeds contain reduced levels of delta-cadinene synthase
a key enzyme in gossypol production without affecting the
enzymes production in other parts of the plant where
gossypol is important in preventing damage from plant
pests.

slide 20:

Biotechnology Agriculture
 Similar efforts have been directed toward the reduction of
the cyanogenic natural product linamarin in cassava
plants.
 Although no plant products that use RNAi-based genetic
engineering have yet passed the experimental stage
development efforts have successfully reduced the levels
of allergens in tomato plants and decreased the
precursors of likely carcinogens in tobacco plants.
 Other plant traits that have been engineered in the
laboratory include the production of non-narcotic natural
products by the opium poppy resistance to common plant
viruses and fortification of plants such as tomatoes with
dietary antioxidants.

slide 21:

RNA interference
characteristics
 dsRNA needs to be directed against an exon
not an intron in order to be effective
 Homology of the dsRNA and the target
gene/mRNA is required
 Targeted mRNA is lost degraded after RNAi
 The effect is non-stoichiometric small
amounts of dsRNA can wipe out an excess of
mRNA pointing to an enzymatic mechanism
 ssRNA does not work as well as dsRNA

slide 22:

Advantage of RNAi
 Downregulation of gene expression simplifies
"knockout" analysis.
 Easier than use of antisense oligonucleotides.
siRNA more effective and sensitive at lower
concentration.
 Cost effective
 High Specifity
middle region 9-14 are most sensitive
 With siRNA the researcher can simultaneously
perform experiments in any cell type of interest
 Can be labelled
 Ease of transfection by use of vector

slide 24:

http://www.rnaiweb.com/RNAi/RNAi_Web/

slide 25:

http://www.rnainterference.org/Sequences.html

slide 26:

RNAi Glossary
 Dicer – Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded
RNAs. Dicer processes long dsRNA into siRNA of 21-23 nt.
 Interferon – A small and highly potent molecule that functions in an autocrine and paracrine manner
and that induces cells to resist viral replication. This term is related to RNAi because in mammals
introduction of dsRNA longer than 30 nt induces a sequence-nonspecific interferon response.
 Micro-RNA – Micro-RNAs miRNA are single-stranded RNAs of 22-nt that are processed from 70-nt
hairpin RNA precursors by Rnase III nuclease Dicer. Similar to siRNAs miRNAs can silence gene
activity via destruction of homologous mRNA in plants or blocking its translation in plants and animals.
 Post-Transcriptional Gene Silencing – Post-transcriptional gene silencing PTGS is a sequence-
specific RNA degradation system designed to act as an anti-viral defense mechanism. A form of PTGS
triggered by transgenic DNA called co-suppression was initially described in plants and a related
phenomenon termed quelling was later observed in the filamentous fungus Neurospora crassa
 Ribozyme – Ribozymes are RNA molecules that act as enzymes in the absence of proteins.
 RNA Interference – RNA Interference RNAi a term coined by Fire et al in 1998 is a phenomenon that
small double-stranded RNA referred as small interference RNA or siRNA can induce efficient
sequence-specific silence of gene expression.
 RNA-Directed DNA Methylation – RNA-directed DNA methylation RdDM is an RNA directed
silencing mechanism found in plants. Similar to RNA interference RNAi RdDM requires a double-
strand RNA that is cut into short 21-26-nt fragments. DNA sequences homologous to these short RNAs
are then methylated and silenced.
 RNA-Induced Silencing Complex – RNA-induced silencing complex RISC is an siRNA-directed
endonuclease catalyzing cleavage of a single phosphodiester bond on the RNA target.
 RNAi Trigger – RNAi triggers are double-stranded RNAs containing 21-23 nt sense and antisens
strands hybridized to have 2 nt overhangs at both 3 ends.
 Small Interfering RNA – Small Interfering RNA siRNA is 21-23-nt double-strand RNA. It guides the
cleavage and degradation of its cognate RNA.
 Helicase – Enzyme responsible for unwinding double stranded molecule